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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for the treatment of cancer because it targets tumor cells while sparing normal cells. Several cancers, however, fail to respond to TRAIL's antineoplastic effects. These resistant tumors require cotreatment with sensitizing agents in order for TRAIL to exert anticancer activity. Histone deacetylase inhibitors (HDACi) have been recognized as potent TRAIL sensitizers. In searching for the determinants of TRAIL responsiveness, HDACi-mediated TRAIL sensitization has been predominantly attributed to TRAIL receptor upregulation. This explanation, however, has been challenged by a few studies. The aim of the present study was to explore the relevance of TRAIL receptor expression for HDACi-mediated TRAIL sensitization in childhood tumors, i.e., in medulloblastoma, Ewing's sarcoma and osteosarcoma. In previous studies, we had shown that TRAIL and HDACi were synergistic in inducing apoptosis in medulloblastoma and Ewing's sarcoma. In the present study, we demonstrate that HDACi cooperated with TRAIL in eliciting cell death in osteosarcoma. However, HDACi treatment did not alter or even reduced cell surface expression of TRAIL receptors in the three childhood tumors. In gaining insight into the apoptotic pathway involved in TRAIL sensitization, HDACi were found to potentiate TRAIL-induced caspase-8 activation. Taken together, our findings suggest that HDACi-mediated TRAIL sensitization is not the result of TRAIL receptor upregulation, but the result of a receptor-proximal event in childhood tumor cells.

Abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for cancer treatment since it provokes cell death in most tumor cells while leaving most normal cells unscathed. Some cancers, however, show resistance to TRAIL, indicating that TRAIL alone may be insufficient for cancer therapy. Here we studied whether the apoptotic susceptibility of A549 non-small cell lung cancer cells could be modulated by inhibiting protein kinase C (PKC). We show that an inhibitor with preference for novel PKC isozymes, NPC 15437, significantly augmented TRAIL sensitivity of A549 cells, as judged by assessing cell death and mitochondrial membrane potential. Likewise, NPC 15437 also significantly potentiated the responsiveness of DAOY medulloblastoma cells to TRAIL. In contrast, an inhibitor with preference for conventional PKC isozymes, Gö6976, did not augment TRAIL sensitivity of A549 cells. To further specify the PKC isozyme responsible for TRAIL sensitization, we used a peptide inhibitor with selectivity for the novel PKC isozyme ɛ, myr-PKCɛ V1-2. The inhibition of PKCɛ resulted in a significant amplification of the cytotoxic activity of TRAIL in A549 cells. Altogether, our study provides evidence for a considerable role of PKCɛ in the apoptotic responsiveness of A549 lung cancer cells, and possibly other malignancies, to TRAIL.

Summary Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and histone deacetylase inhibitors (HDIs) show promise for the treatment of cancer. However, in a number of reports they have been proven ineffective to induce cell death when applied as single agents. In this study, we show that A549 lung carcinoma cells and PC-3 prostate carcinoma cells underwent substantial apoptosis when coexposed to TRAIL and either suberoylanilide hydroxamic acid, sodium butyrate or trichostatin A. HDIs and TRAIL synergized in activation of capase-3, induction of internucleosomal DNA fragmentation and promoting mitochondrial damage. Significantly, cotreatment with minimally toxic doses of HDIs and TRAIL resulted in a marked apoptotic response in both cell lines. These data provide a rationale for a more in-depth exploration into the potential of combining TRAIL and HDIs as a valuable anticancer strategy.

Anthracyclines such as doxorubicin remain among the most effective agents for the treatment of solid tumors and hematological malignancies. To overcome dose-limiting side effects like cardiotoxicity, an intensive effort has been undertaken to develop promising doxorubicin prodrugs that are specifically activated at the tumor site. One approach is the application of peptide prodrugs of doxorubicin. The enzyme cathepsin B catalyzes the activation of these prodrugs, and hence, the regulation of cathepsin B by antitumor agents could influence the efficacy of peptide prodrugs using this protease. In the present investigation, the effects of doxorubicin on cathepsin B expression in the human cervix carcinoma cell line HeLa were examined. Exposure to doxorubicin induced a time- and dose-dependent up-regulation of cathepsin B expression on mRNA, protein, and activity levels. In the cathepsin B gene promoter region, a potential nuclear factor κB (NF-κB) binding site could be identified. Pretreatment of HeLa cells with specific NF-κB inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein IκB could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicin-induced cathepsin B expression. Finally, binding of NF-κB subunits p50 and p65 to the NF-κB binding site in the cathepsin B gene promoter region could be demonstrated by electrophoretic mobility shift assay. In summary, our data clearly indicate that doxorubicin induces cathepsin B expression and activity via NF-κB. These findings contribute to a better understanding of tumor targeting with peptide prodrugs and help to define a possible mechanism of doxorubicin toxicity in tumor cells.

We investigated the cytotoxic responsiveness of 40 cell lines derived from representatives of the Ewing's sarcoma family of tumours (ESFT), i.e., Ewing's sarcoma (ES), peripheral primitive neuroectodermal tumour (pPNET) and Askin tumour (AT), to tumour necrosis factor–related apoptosis-inducing ligand (TRAIL). Incubation with TRAIL at 100 ng/ml induced cell death at 24 hr in 19 of 26 ES, 11 of 12 pPNET and 2 of 2 AT cell lines. Half-maximal cell death concentrations (IC50 values) varied from 0.1 to 20 ng/ml. TRAIL displayed potent cytotoxic activity against freshly derived ESFT cell isolates. Cytotoxicity was associated with phosphatidylserine expression and internucleosomal DNA fragmentation, features characteristic of apoptosis. The apoptotic programme in the sensitive ESFT VH-64 cell line revealed TRAIL-induced activation of FLICE/MACH1 (caspase-8) and CPP32/Yama/apopain (caspase-3) and processing of the prototype caspase substrate poly(ADP-ribose) polymerase. In addition, TRAIL provoked a collapse of the mitochondrial transmembrane potential (ΔΨm), parallelled by a reduction in ATP levels and release of cytochrome c from mitochondria into the cytosol. Inhibition of caspase-8 and caspase-3 by zIETDfmk and zDEVDfmk, respectively, substantially prevented TRAIL-induced apoptosis. However, zIETDfmk, but not zDEVDfmk, reduced TRAIL-mediated ΔΨm dissipation, indicating that TRAIL causes mitochondrial dysfunction through caspase-8 acting upstream of mitochondria. While macromolecule synthesis inhibitors (actinomycin D, cycloheximide) augmented susceptibility to TRAIL in TRAIL-responsive cell lines, these agents did not render TRAIL-resistant cell lines susceptible to TRAIL. However, the proteasome inhibitor MG132 sensitised to TRAIL in resistant cell lines. Collectively, these results show that TRAIL initiates effective death in the vast majority (80%) of cell lines derived from ESFT. Since TRAIL provoked cell death in ESFT ex vivo, this cytokine may be a promising drug for the treatment of ESFT in vivo. Int. J. Cancer 88:252–259, 2000. © 2000 Wiley-Liss, Inc.

cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233–238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura 2–dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.

The cAMP signaling cascade leading to changes in [Ca2+]iin Dictyostelium discoideumwas analyzed using cell lines overexpressing single cAMP receptor subtypes (cAR1–cAR3) or lacking the Gα2or Gβsubunit of the G protein. Imaging of fura2‐dextran‐loaded amoebae revealed cAMP‐induced [Ca2+]ichanges characteristic for each receptor subtype activated. Cells expressing distinct subtypes sort to defined zones during multicellular development suggesting involvement of the specific [Ca2+]itransients in patterning processes. Whereas generation of the [Ca2+]iincrease was Gα2‐independent, only few cells devoid of Gβdisplayed a [Ca2+]ichange after stimulation indicating its participation in the regulation of the calcium homeostasis.

We have previously shown that the Dictyostelium discoideumribosomal protein L19 specifically binds Ca2+/calmodulin [Sonneman et al. (1991) J. Biol. Chem. 266, 23091‐23096]. To investigate the role of calmodulin in the regulation of protein synthesis, we have now established an in vitro protein synthesizing system from Dictyosteliumcells which can elongate polypeptide chains with high efficiency. Various calmodulin antagonists affected translation in this system. The inhibitory effects of the antagonists could be partially reversed by addition of calmodulin. A monoclonal antibody against D. discoideumcalmodulin also specifically inhibited protein synthesis. Similar effects of calmodulin antagonists were found in a standard wheat germ in vitro translation system.